Fig. 5: SHANK3 depletion impairs the growth of pre-existing KRAS-mutant PDAC tumours.
From: SHANK3 depletion leads to ERK signalling overdose and cell death in KRAS-mutant cancers
a A schematic representation of the lentiviral vector for tetracycline/doxycycline (dox)-inducible synthesis of SHANK3 shRNA with a tRFP (TurboRFP) reporter for visual confirmation of shRNA expression following dox induction. 5’LTR, 5’long terminal repeat; Ψ, Psi packaging sequence; PuroR, puromycin resistance gene; 2a, self-cleaving peptide; WPRE, Woodchuck Hepatitis Post-transcriptional Regulatory Element; 3’ SIN LTR, 3’ Self-inactivating Long Terminal Repeat (see methods for more detail). b ERK activation kinetics in shSHANK3 KRAS-mutant cells. Representative immunoblots showing SHANK3, p-ERK and cleaved-PARP1 levels in control (-dox) and dox-induced (+dox) shSHANK3-expressing PANC-1 cells (mix of two independent clones) collected at different time points. GAPDH, loading control (n = three independent experiments). c–e Analysis of the growth and viability of shSHANK3-expressing PANC-1 spheroids ± dox (dox added at day 5, when spheroids were established, and continued until day 15). Representative images show SHANK3 depletion as observed by the tRFP reporter and apoptotic Annexin V-positive cells (c). Quantification of spheroid growth over time (d), shaded region denotes sphere growth prior to treatment and cell viability at endpoint (e) (mean ± s.d. from n = 3 independent experiments; unpaired two-tailed Student’s t-test with Welch’s correction at endpoint). f, g Caspase-3 (f) and caspase-8 activity (g) in shSHANK3-expressing PANC-1 cells ± dox at the indicated time points (shown is normalized fluorescence intensity). Staurosporine used as a positive control (mean ± s.d.; n = 3 independent experiments; one-way ANOVA with Holm-Sidak’s multiple comparison test). h–l Analysis of the growth of established tumours in mice following SHANK3 depletion. h Outline of animal experiments. i Tumour volumes after starting the dox treatment (normalised to tumour volumes at the start of dox induction). j Representative IVIS images of the tRFP reporter expression in tumours 5 and 26 days after dox induction. k SHANK3 gene expression (mRNA levels) in tumours at the end of the experiment. l Tumour weights at the end of the experiment (26 days after dox-induction) (data represent individual tumours and the mean ± s.d.; n = 11 (-dox) and 12 (+dox) tumours; unpaired Student’s t-test with Welch’s correction). Source data are provided as a Source Data file.
